Corresponding author: Oleg E. Kosterin ( firstname.lastname@example.org )
Academic editor: Vladimir Lukhtanov
© 2015 Vladimir I. Solovyev, Yuri Ilinsky, Oleg E. Kosterin.
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Citation: Solovyev VI, Ilinsky Y, Kosterin OE (2015) Genetic integrity of four species of Leptidea (Pieridae, Lepidoptera) as sampled in sympatry in West Siberia. Comparative Cytogenetics 9(3): 299-324. doi: 10.3897/CompCytogen.v9i3.4636
In southern West Siberia, as many as four Leptidea Billberg, 1820 species are present sympatrically: L. amurensis (Ménétriés, 1859), L. morsei (Ménétriés, 1859), L. sinapis (Linnaeus, 1758) and L. juvernica Williams, 1946. The two latter were recently recognised as nearly sibling species on morphological and molecular characters. Specimens intermediate as to their subtle diagnostic characters occurring in West Siberia and elsewhere were interpreted as resulted from limited introgression. This supposition was tested via populational morphological and molecular analysis of spring brood specimens of all the four species taken from a limited (4.5 × 0.2 km) area in the suburbs of Novosibirsk. The samples were analysed with respect to the genitalic morphology, external characters, three nuclear (CAD, H1 gene and ITS2) and one mitochondrial (COI) molecular markers, infection of the intracellular maternally inherited bacterial symbiont Wolbachia Hertig, 1836 and its wsp gene coding for a hypervariable surface protein. Interspecific variation of the nuclear CAD and ITS2 sequences and the mitochondrial COI gene in L. sinapis and L. juvernica turned out concordant. The absence of molecular evidence of introgression suggests genetic integrity of these two species and allows their reliable identification by molecular characters. The genitalic (lengths of the saccus and valva) and external characters (wing pattern) of males overlap in L. sinapis and L. juvernica, as identified by molecular markers and thus are not so helpful in actual species identification. Only the ductus bursae length showed no overlap and can be used for identification of females. The histone H1 gene appeared five times less variable over the four studied species than COI, and found to be identical in species L. sinapis and L. juvernica. Wolbachia infection was found in all studied species. We identified three wsp variants of Wolbachia: 1) wsp-10 allele in L. amurensis, L. sinapis, L. juvernica; 2) a very similar wsp-687 allele in L. sinapis; and 3) wsp-688, highly divergent to the previous ones, in L. morsei.
Leptidea, Lepidoptera, Wolbachia, introgression, molecular markers, histone H1, COI, ITS2, wsp, genitalia morphology, intraspecific variation
The genus Leptidea Billberg, 1820 (Dismorphiinae, Pieridae) includes several (at least eight) Palearctic species. Recently it attracted attention because of repeated and rather unexpected discoveries of sibling species (
The main diagnostic character of L. reali and L. juvernica versus L. sinapis is a substantially greater relative lengths of the aedeagus and saccus (which correlate to each other) in the male genitalia and of the ductus bursae in the female genitalia (
spring brood males of L. juvernica have in general darker, more suffused hind wing underside below vein M3 and with less distinct stripy pattern (less expressed lighter postdiscal spots between veins) than those of L. sinapis;
summer brood males of L. juvernica differ from those of L. sinapis in the fore wing upperside without the light rim along the apical dark spot and darkened ends of veins M3 and Cu1;
in West Siberia, the spring brood males of L. juvernica were claimed to have a more attenuated fore wing apex than those of L. sinapis (
No external differences were revealed between females of the two species.
Authors working in different regions (
These facts can be interpreted in three ways: (i) as resulting from some gene exchange (introgression) between L. sinapis and L. juvernica; (ii) as common polymorphism of genes affecting the genitalia structure and/or wing coloration, inherited from the common ancestor, or (iii) by independent mutations (homoplasy) of these hypothetical genes.
The relationships of closely related species may be clarified via two approaches, the phylogeographic and population genetic ones. The former approach implies accumulation of data from a territory as broad as possible in order to reconstruct the history of divergence and spread of species. The latter approach consists of analysing large samples from certain populations in order to register phenomena such as deviations from panmixia, linkage disequilibrium, gene exchange between sympatric taxa, and effects of natural selection.
Relationships between sibling species of Leptidea were mostly studied via the phylogeographic approach applied to the entire species ranges (
Another approach is searching for particular mechanisms of isolation between these two species. Hybridisation experiments revealed that prezygotic isolation between L. sinapis and L. juvernica or L. reali was probably based on behavioral barriers, for instance recognition by females of a species-specific courtship behaviour of males or species-specific pheromones (
The western foothills of the Altay-Sayan Mountain System (West Siberia, Russia) are unique in being inhabited by four Leptidea species altogether, more than elsewhere in the world: Leptidea morsei (Fenton, 1881), L. amurensis (Ménétriés, 1859), L. sinapis and L. juvernica (Fig.
Spring brood males of four species of Leptidea Billberg, 1820: L. amurensis (Ménétriés, 1859) (a), L. morsei (Fenton, 1881) (b), L. sinapis (Linnaeus, 1758) (c) and L. juvernica Williams, 1946 (d), simultaneously collected in the studied area at the border of Novosibirsk city and Berdsk town, West Siberia, Russia (after
The main attention was paid to the closely related and supposedly hybridising species L. sinapis and L. juvernica. They were analysed with respect to the popular mitochondrial marker COI (the gene for cytochrome oxidase I) and the nuclear markers CAD (the gene for carbamoyl phosphate synthetase II, aspartate carbamoyltransferase, dihydroorotase), ITS2 (internal transcribed spacer 2 in the ribosome cluster), and the histone H1 gene, designated here as H1. A histone H1 gene was recently proposed as a good phylogenetic marker (
Seventy spring brood specimens of Leptidea spp. were collected in the vicinity of Novosibirsk Academy Town, Novosibirsk Province. The collection area was a 100–200 m wide and a 4.5 km long continuous stripe of meadows adjacent to birch/ pine forests, along the bank of the Novosibirsk Water Reserve and the parallel railroad, between Obskoe More railway station (54°47'37"N; 83°04'34"E; (DMS)) and a point (54°50'04"N; 83°04'40"E (DMS)), 900 m NNE of Rechkunovka railway station, at elevations of 107–137 m a.s.l. (see the locality on a schematic map of northern Eurasia in Fig.
Position (black circle) of the studied area at the border of Novosibirsk city and Berdsk town (54°47'37"N; 83°04'34"E – 54°50'04"N; 83°04'40"E; DMS), Novosibirsk Province, Russia, on a schematic map of northern Eurasia.
Material collected, COI gene allelic states as revealed by CAPS approach (denoted as follows: s – Leptidea sinapis, j – L. juvernica, a – L. amurensis, m – L. morsei), European Nucleotide Archive (ENA) accession numbers of the COI and H1 gene sequences, presence of Wolbachia infection (+ detected; - not detected) and the wsp alleles according to the PubMLST database.
|Specimen||Sex||Date collection||COI variant||COI ENA accession number||H1 ENA accession number||
Genomic DNA was extracted according to
A 708 bp long fragment of the COI gene, positions 1526–2156 (positions are given according to the mitochondrial reference of Drosophila yakuba Burla, 1954 (AN X03240)), was amplified with the universal insect primers LCO-1490 and HCO-2198 (
DNA samples were examined for Wolbachia infection by amplification of wsp with the following primer set: wsp81F (5’TGGTCCAATAAGTGATGAAGAAAC-3’), wsp691R (5’AAAAATTAAACGCTACTCCA-3’) (
PCR mixtures (30 μl) contained 0.2 mM of each dNTP, 1.5 mM MgCl2, 25mM KCl, 60 mM Tris-HCl (pH 8.5), 10 mM β-mercaptoethanol, 0.1% Triton X-100, 0.5 µM of each primer, 1 μl of genomic DNA solution and 1 U of Taq DNA polymerase or 1 U of Smart-Taq DNA Polymerase (by Laboratory Medigen, Novosibirsk, Russia). PCR was performed using a thermal cycler MyCycler (Bio-Rad, USA) with the following program: 1) 94 °C — 2 min 30 s, 1 cycle; 2) 95 °C — 15 s, 47–55 °C — 30 s, 68 °C — 1 min, 35 cycles; 3) 68 °C — 2 min, 1 cycle.
The entire coding sequence of H1 and a 631 bp long fragment of COI (positions 1526–2156) were sequenced. The Sanger reaction was conducted in 30 μl volume of mixture containing 1 μl of BigDye Terminator, version 3.1 (Applied Biosystems), 100–200 ng of DNA, 3 pmol of primer and 6 μl of buffer solution for BigDye 3.1. A MyCycler (Biorad) thermocycler was used with the following program: 95 °C — 45 s, 50 °C — 30 s, 60 °C — 4 min; 26 cycles. Sequencing was made at the SB RAS Genomic Core Facility, Novosibirsk.
Sequence alignments and calculation of the genetic distances were performed using the MEGA 5.0 software package (
For genotyping the L. sinapis and L. juvernica specimens with respect to certain diagnostic nucleotide substitutions in mitochondrial and nuclear markers, CAPS analysis was conducted (
The 708 bp long amplified fragment of COI of L. juvernica contains three restriction sites for endonuclease HpaII and is digested to 4 fragments (66, 109, 206, 327 bp), while the orthologous fragment of L. sinapis has no restriction sites. The ITS2 region of L. sinapis contains the only site specific for endonuclease AluI and is digested into 2 fragments (412, 272 bp); the ITS2 of L. juvernica does not contain restriction sites for AluI. The CAD sequence of L. juvernica includes only one restriction site for endonuclease HindIII, which digests it into two fragments, 110 and 461 bp in length; the CAD sequence of L. sinapis has two sites which produce three digestion fragments (110, 189, 272 bp). The buffers and enzymes for restriction reactions were produced by Sibenzim, Novosibirsk, Russia. The identical procedure was used for different markers, as follows: 9 μl of the PCR product was added with 0.5 U of endonuclease and 1 μl of a buffer relevant to the endonuclease. The mixture was incubated at 37 °C for 2 hr, inactivated at 80 °C for 20 min and analyzed by electrophoresis in 1.5% agarose.
The abdomen tip with the genitalia was taken from frozen specimens of L. juvernica and L. sinapis, incubated for 10 min at 98 °C in 10% potassium hydroxide for maceration and dissected under a stereomicroscope. Lengths of the valve (V) and saccus (S) were measured with an ocular-micrometer and binocular lens MBS-2, as shown in Fig.
Male (a specimen L12) and female (b specimen L45) genitalia of Leptidea juvernica, with measured parameters indicated, namely the length of the saccus (S), valve (V) and ductus bursae (D).
Statistical analyses were carried out using MS Excel 10 for Windows.
The genitalia were analysed before molecular analysis, which was carried out blindly of the genitalic results. The specimens in which molecular results appeared discordant with morphological ones, were then rechecked for morphology and discordancy was confirmed.
The external characters reported to be different in the spring brood males of L. juvernica and L. sinapis, namely (1) the wing underside below vein M3 more suffused by dark scales and with less expressed lighter spots between veins and (ii) more attenuate fore wing apex in the former species (
hind wing underside suffusion below vein M3: 0 – with well-expressed lighter spots between veins, 1 – stronger, more even, with scarcely or expressed lighter spots;
fore wing apex: 0 – broadly rounded; 1 – more attenuated and acute (Fig.
Males of Leptidea sinapis (a specimen L1 b specimen L26) and Leptidea juvernica, (c specimen L3 d specimen L8), with different scores of subjectively evaluated wing characters: the hind wing underside suffusion below vein M3: 0 – lighter, with better expressed lighter spots between veins; 1 – stronger, with scarcely seen lighter spots; and the fore wing apex shape: 1 – more acute, 0 – more rounded. The scores for the shown specimens are as follows (suffusion, apex shape): a (0,0); b (1,0); c (1,1); d (1,0); a and c are variants most frequent in the respective species.
The 631 bp long fragment of the mitochondrial gene COI (position 1526 – 2156) was sequenced for 34 Leptidea specimens (10 of L. sinapis + L. juvernica, 11 of L. morsei and 13 of L. amurensis) collected in the same locality. The sequences were submitted to European Nucleotide Archive (ENA), for accession numbers see Table
Polymorphic positions in the COI gene in Leptidea sinapis (specimens L15-L18) and L. juvernica (specimens L10–L14 and L19). Positions discriminating s- and j- allele types are boldfaced; intraspecific substitutions are underlined.
|Specimens representing six alleles||L10, L11, L14||L12, L13||L19||L15||L16, L17||L18|
The averaged and minimum p-distances between of the studied COI fragment between L. juvernica (j-alleles), L. sinapis (s-alleles) L. morsei, L. amurensis, of are provided in Table
Evolutionary Divergence over Sequence Pairs between Leptidea species in the studied sample as calculated from 34 COI gene sequences obtained. The number of nucleotide substitutions per site averaged over all possible specimens pairs for any two species, ± its standard error, is shown below the main diagonal, their minimum value among all specimens pairs for any two species is shown above the main diagonal. The total number of positions was 631.
|1. L. juvernica||0.024||0.041||0.052|
|2. L. sinapis||0.029±0.006||0.043||0.052|
|3. L. morsei||0.043±0.008||0.043±0.008||0.048|
|4. L. amurensis||0.055±0.008||0.053±0.008||0.048±0.008|
PCR amplification of the wsp gene revealed Wolbachia infection in 38 of 42 tested males and 18 of 19 tested females of the L. sinapis + L. juvernica united sample (91.8% prevalence), in all 11 tested specimens of L. morsei and in all 13 tested specimens of L. amurensis (100% prevalence) (Table
The wsp gene was sequenced in one specimen of each L. amurensis (L58), L. morsei (L67) and L. juvernica (L12) and two specimens of L. sinapis (L16, L17). The sequences were submitted to the PubMLST database http://pubmlst.org [accessed 30 January 2015] (for accession numbers see Table
The CAPS approach (see ‘Materials and methods’) allowed us to test 36 more specimens of L. sinapis/L. juvernica in addition to those 10 in which COI was sequenced. We distinguished s- versus j-alleles of the mitochondrial marker COI and nuclear markers CAD and ITS2 in the same set of specimens. The three sets of CAPS data, for all three markers, were fully concordant: each specimen possessed either only s- or only j-alleles for all three markers. This gave us a reason to consider and further refer these specimens as belonging to the true biological species L. sinapis and L. juvernica, respectively.
The 747 bp long coding sequence of the H1 gene of histone H1 was sequenced in 9 specimens: L10, L12 (L. juvernica), L15–L17 (L. sinapis), L50, L58 (L. amurensis), L60, L67 (L. morsei); the sequences were submitted to ENA (for the accession numbers see Table
The lengths of the following genital structures were measured: the saccus and valve in males (Tables
The genital measurements of the studied samples of Leptidea spp. The mean values and standard deviations are given of the lengths for the saccus (S), valve (V) and their ratio (S/V) in the male genitalia and the length of the ductus bursae (D) in the female genitalia in the studied samples of Leptidea sinapis and L. juvernica, as identified by molecular markers, and the united sample of both species.
|standard deviation||L. juvernica||0.10||0.07||0.15||0.16|
|T-criterion for differentiation between the species||5.60||2.84||7.49||9.42|
|significance||P < 0.001||P < 0.01||P < 0.001||P < 0.001|
CAPS-analysis data, the lengths of the saccus (S), valve (V) and their ratio (S/V) in males of L. sinapis and L. juvernica.
|COI/ HpaII||ITS2/ AluI||CAD HindIII||
CAPS-analysis data and the lengths of the ductus in females of L. sinapis and L. juvernica.
|The length of ductus (mm)|
|COI/ HpaII||ITS2/ AluI||CAD/ HindIII|
Additional characters: the saccus curvature, general size, hind wing underside suffusion and fore wing apex shape, classified to arbitrary classes, in the studied male specimens of Leptidea sinapis and L. juvernica. Character states: saccus: 0 – straight, 1 – S-like curved; general size: 0 – large, 1 – small; hind wing underside suffusion below vein M3: 0 – with well-expressed lighter spots between veins, 1 – rather even, with very scarcely or not expressed lighter spots; fore wing apex: 0 – broadly rounded; 1 – more attenuated and acute. The typical L. sinapis phenotype corresponds to the character states 0000, the typical L. juvernica to 1111.
|Specimen||Molecular identification||Saccus curvature||General size||Hind wing underside suffusion||Fore wing apex|
Two classes of spring brood females of the s- and j-groups with respect to the ductus bursae length were concordant with the CAPS data. The mean ductus length was significantly (p<.001) inferior in the s-group, and the length distributions of these groups did not overlap (Tables
The saccus and valve lengths of the L. sinapis and L. juvernica. A Plot of the saccus length against the valve length of L. sinapis and L. juvernica, as identified by molecular markers B Plot of the ratio of the saccus length to the valve length against the saccus length for the same sample.
The saccus curvature did not appear as a reliable differentiating character as well, since its mean square contingency coefficient (φ coefficient) value with the CAPS data was rather small (φ = 0.50, p<.001).
The size, coloration of the hind wing underside and the shape of apex of the fore wing were also found to associate with the molecular groups j and s but again with small values of the φ coefficient: φ = 0.49 for the size, φ = 0.73 for the hind wing coloration p<0.001; φ = 0.29 for the fore wing apex p<0.05.
It may be concluded that neither the genital structure lengths, nor the saccus curvature, nor the general size, nor the wing coloration allow reliable identification of males of the s- and j-groups.
The observed differences in the studied COI fragment of L. sinapis and L. juvernica are substantial. They are illustrated by the averaged and minimum p-distances provided in Table
In some studies the task of quick and still reliable identification of species of the L. sinapis complex by application of a morphometric approach was achieved with a 100% efficiency (
According to the discriminant criterion suggested by
The length of the saccus and valve in our case appeared insufficient for a complete discrimination of L. sinapis and L. juvernica. The ratio of these values, which allowed 98% discrimination of males of L. sinapis and L. reali (
Other external characters, such as the general size and coloration of the hind wing underside, recognised by a naked eye, are unreliable and allow only a first approach to species identification in the field (
Anyway, we conclude that molecular and karyological characters are so far the only reliable means of identification of L. sinapis and L. juvernica, and the molecular ones are much easier methodically.
Divergence and fixation of alleles of genes responsible for reproductive isolation are sufficient for speciation (
The overlap of distribution of the length of the male genital structures may be interpreted through presence in both species of both ‘long’ and ‘short’ alleles of the hypothetical gene responsible for differences between L. sinapis and L. juvernica, although with oppositely biased frequencies. This could result from:
inheritance of both alleles from the common ancestor,
introgression between species, and
de novo mutational re-appearance of ‘long’ and/or ‘short’ alleles.
Introgression is a common phenomenon for sympatric closely related butterfly species. According to an estimation by
Specimens from Novosibirsk Province with intermediate state of diagnostic external characters or, more frequently, with discordant combination of characters of L. sinapis and L. juvernica were supposed to be interspecies hybrids or products of their backcrosses (
An attempt to reveal hybridization between L. sinapis and L. reali in the Pyrenees using 16 allozyme loci was unsuccessful (
Inheritance of ‘long’ and ‘short’ alleles in the common ancestor of L. sinapis and L. juvernica is also a plausible interpretation. These alleles could be involved in genetic isolation of the nascent species by forming a reproductive barrier between them, but fixation of either allele in these species may not have taken place. The initial genital prezygotic barrier could later be strengthened by adding biochemical and behavioral barriers. They would lower significance of the primary genital barrier and somewhat release selection for the ‘long’ versus ‘short’ alleles and vice versa, allowing their frequency to drift.
At this stage of our knowledge, the third scenario of arising ‘long’ and/or ‘short’ allele(s) cannot be excluded as well.
In contrast to core histones, histone H1 is a very variable protein (
All the four studied Leptidea species were found to be infected with Wolbachia, with prevalence of infected specimens of 91.8% in L. sinapis + L. juvernica and 100% in L. amurensis and L. morsei (Table
Wolbachia infection is vertically transmitted through host generations via maternal cytoplasm. Therefore the phylogeny of Wolbachia could be expected to be concordant with the phylogeny of its hosts. However, Wolbachia can as well be transmitted horizontally between related species through introgression and between unrelated species by unknown agents. In addition, Wolbachia strains as well as their particular genes such as wsp may result from recombination between different strains.
We found three Wolbachia strains in Leptidea according to the wsp gene sequences. Three species, L. amurensis, L. sinapis and L. juvernica, were found to possess allele wsp- 10 which is widespread in insects. According to our counts at the http://pubmlst.org, it was so far registered in 27 species of Lepidoptera from different families (Pyralidae, Hesperiidae, Papilionidae, Pieridae, Nymphalidae, Lycaenidae) and, and also in Culex pipiens Linnaeus, 1758 from Diptera. The second allele wsp-687 was found in L. sinapis for the first time. It differs from wsp-10 in only one nucleotide substitution. The third wsp-688 allele was also found for the first time, in L. morsei. This new allele has a unique hypervariable region 2, HVR2-267, while other hypervariable regions HVR1-2, HVR3-2, HVR-23 were found elsewhere (
The pattern of Wolbachia variants in the studied Leptidea species is discordant to the host phylogeny. Variation of wsp sequences in L. sinapis, L. juvernica and L. amurensis is extremely low, viz. wsp-10 allele is common for these species and a closely related wsp-687 allele is also found in L. sinapis, whereas L. morsei possesses a highly divergent wsp-688 allele.
The wsp-10 allele could hardly be inherited from the common ancestor of the three species, taking into account a considerable degree of variation accumulated by the host Leptidea genes, both nuclear and mitochondrial (
the same strain of Wolbachia could have spread across the three species by interspecies crosses;
The same wsp allele could have spread across the three species via horizontal transfer of Wolbachia.
We exclude option (i) since we failed to trace such crosses by other molecular means. Explanation (ii), that is independent infection by the same Wolbachia strain, is most probable because of a high frequency of wsp-10 in butterflies. L. morsei was no doubt independently infected by an unusual Wolbachia strain with wsp-688, however, more data on L. morsei is necessary to consider the evolutionary history of its Wolbachia.
Leptidea amurensis, L. morsei, L. sinapis and L. juvernica coexist in the same locality in West Siberia without detectable introgression. Each of the molecular characters COI, CAD and ITS2 markers, as well as the length of the female ductus bursae, allow a reliable identification of L. sinapis and L. juvernica. The length of the saccus related to that of the valva as the most easily assessed male genitalic character, as well as the characters of wing pattern and shape in males, are unreliable for identification of these two species. An overwhelming majority of Leptidea individuals are infected with Wolbachia. Three alleles of the Wolbachia gene wsp were recorded (two of them for the first time), that of L. morsei being highly divergent from the allele found in L. amurensis, L. juvernica and L. sinapis (this species contains a very similar third allele), which is discordant with the presumed phylogeny of the host.
The work was supported by Russian State project VI.53.1.3. and the project 13-04-00516a by the Russian Fund for Fundamental Research. We are grateful to Martin Wiemers and an anonymous reviewer for numerous valuable comments to the initial version of the paper.